High levels of cyclic di-GMP in Klebsiella pneumoniae attenuate virulence in the lung

ABSTRACT The bacterial second messenger bis-(3′-5′)-cyclic dimeric GMP (c-di-GMP) has been shown to influence the expression of virulence factors in certain pathogenic bacteria, but little is known about its activity in the increasingly antibiotic-resistant pathogen Klebsiella pneumoniae . Here, the expression in K. pneumoniae of a heterologous diguanylate cyclase increased the bacterial c-di-GMP concentration and attenuated pathogenesis in murine pneumonia. This attenuation remained evident in mice lacking the c-di-GMP sensor STING, indicating that the high c-di-GMP concentration exerted its influence not on host responses but on bacterial physiology. While serum resistance and capsule expression were unaffected by the increased c-di-GMP concentration, both type 3 and type 1 pili were strongly upregulated. Importantly, attenuation of K. pneumoniae virulence by high c-di-GMP levels was abrogated when type 1 pilus expression was silenced. We conclude that increased type 1 piliation may hamper K. pneumoniae virulence in the respiratory tract and that c-di-GMP signaling represents a potential therapeutic target for antibiotic-resistant K. pneumoniae in this niche. </jats:p

Growth delay in small EMT6 spheroids induced by cytotoxic drugs and its modification by misonidazole pretreatment under hypoxic conditions.

Experiments have been carried out to determine whether the growth delay induced in small EMT6 spheroids (approximately 250 micrometers in diameter) by a range of cytotoxic drugs can be increased by pre-incubation of the spheroids under hypoxic conditions with misonidazole (MISO). Hypoxic pre-incubation was for 3 or 5 h in the presence of 5 mM MISO, and caused a growth delay of about 1 day or 2 days respectively. Sensitization to nitrogen mustard (HN2), melphalan, chlorambucil, BCNU and CCNU was seen, but the shapes of the dose-response curves and the ratio of effects for 3 h and 5 h pretreatment varied between the drugs. In contrast to the other agents, hypoxic pre-incubation with MISO reduced the spheroid response to adriamycin